Myxococcus xanthus is an excellent prokaryotic system for studying the regulation of development. M. xanthus show a developmental cycle which is similar to the eukaryotic slime molds such as Dictyostelium discoideum. However, there are several distinct advantages to studying development in M. xanthus, since it is a simple gram-negative bacterium. We propose to study morphogenesis and the molecular mechanisms of control of gene expression during development using M xanthus as a model system. We will continue to work on the on-going projects such as protein S, a development-specific protein, and its gene. We will also attempt to explore several new aspects of M. xanthus, including characterization of multicopy single-stranded DNA (msDNA), a unique DNA found in myxobacteria, the use of anti-sense RNA (micRNA) for characterization of developmental genes, and identification and characterization of specific organelle(s) required for gliding motility. The following are specific aims which we will pursue in this proposal: (1) Development-specific Genes. Two tandemly repeated, homologous genes, ops (protein S-1) and tps (protein S) are differentially expressed during development. Using oligonucleotide-directed site-specific mutagenesis, we will elucidate the exact nucleotide sequences in their promoters which are responsible for the regulation. We will develop a micRNA cloning vector for study of the regulatory mechanisms and the functions of developmental genes isolated. We will also explore cloning the genes for sigma factors for developmental genes. (2) Functions of Protein S and Protein S-1. Protein S-1 will be purified and crystallized, and its biochemical properties will be compared with protein S. Roles of protein S will be further investigated by creating mutations at putative Ca++ binding sites. (3) msDNA. We will determine the RNA primer structures of both msDNAs, from M. xanthus and Sigmatella aurantiaca, and attempt to elucidate the molecular mechanisms of the biosynthesis of msDNA and its role in myxobacteria. (4) Organelles for Gliding Motility. We have isolated microtubule-like structures from M. xanthus. We will characterize these components from biochemical as well as functional aspects. (5) Integration-type Plasmids. We will construct plasmids which facilitate P1 specialized transduction to integrate any genes (or DNA fragments) of interest into the specific site of the M. xanthus chromosome.